Reengineering mRNA primary structure for enhanced protein production

Abstract

Described herein are rules to modify natural mRNAs or to engineer synthetic mRNAs to increase their translation efficiencies. These rules describe modifications to mRNA coding and 3′ UTR sequences intended to enhance protein synthesis by: 1) decreasing ribosomal diversion via AUG or non-canonical initiation codons in coding sequences, and / or 2) by evading miRNA-mediated down-regulation by eliminating one or more miRNA binding sites in coding sequences.

Description

REFERENCE TO PRIORITY DOCUMENT[0001]This application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Application Ser. No. 61 / 155,049, filed Feb. 24, 2009, entitled “Reengineering mRNA Primary Structure for Enhanced Protein Production.” The subject matter of the above-noted application is incorporated by reference in its entirety by reference thereto.INCORPORATION BY REFERENCE[0002]The contents of the text file named “37651-503001WO Sequence listing.txt” which was created on Oct. 26, 2011 and is 53 KB in size, are hereby incorporated by reference in their entirety.BACKGROUND[0003]Translation initiation in eukaryotes involves recruitment by mRNAs of the 40S ribosomal subunit and other components of the translation machinery at either the 5′ cap-structure or an internal ribosome entry site (IRES). Following its recruitment, the 40S subunit moves to an initiation codon. One widely held notion of translation initiation postulates that the 40S subunit moves from...

AI Technical Summary

Benefits of technology

The patent text describes a method for improving the efficiency and stability of protein translation and increasing protein yield and concentration in industrial production of protein drugs. The method involves mutating one or more secondary initiation codons in the coding sequence of a protein to decrease ribosomal diversion away from the primary initiation codon, thereby increasing full-length protein expression efficiency. The method can also involve mutating one or more nucleotides or secondary initiation codons that are located upstream or downstream from a ribosomal recruitment site. Mutating one or more secondary initiation codons can avoid introducing new initiation codons or changing usage bias of mutated codons. The generation of truncated proteins, polypeptides, or peptides other than the full-length encoded protein can be reduced. Mutating one or more secondary initiation codons can also avoid introducing miRNA seed sequences, splice donor or acceptor sites, or mRNA destabilization elements.

Problems solved by technology

However, the notion that translation initiates by a scanning mechanism does not consider the effects of potential initiation codons in coding sequences on protein synthesis.

The negative impact of putative initiation codons in mRNA coding sequences and miRNA-binding sites in mRNAs pose challenges to the pharmaceutical industry.

Poor protein expression limits the large-scale use of certain technologies, for example, problems in expressing enough antigen from a DNA vaccine to generate an immune response to conduct a phase 3 clinical trial.

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